Cell based sensor technologies utilizing electrically active living cells cultured on extracellular electrode arrays have been a focus of research since 1972 (Thomas et al. (1972) Experimental Cell Research 74:61-66). However, most efforts have been directed towards pure neuroscience applications that require experienced interpretation of the signals. More recent work by some groups has attempted to expand the practical uses of action potential (AP) based systems to environmental and chemical monitoring and pharmaceutical screening. However, there has been little work done on signal interpretation models for general classification of agents modulating cellular electrical characteristics.
The membrane capacitance, membrane conductance, cell/substrate separation and action potential parameters of a cell are significant markers regarding a cell""s metabolic state, including general cellular health and ionic channel activity. The membrane potential, the voltage difference across a cell""s plasma membrane, depends on the distribution of ionic charge. For example, in a metallic conductor, the mobile particles carrying charge are electrons; in an aqueous solution, the mobile particles are ions such as Na+, K+, Clxe2x88x92, and Ca++. In an aqueous solution, the number of positive and negative charges are normally balanced exactly, so that the net charge per unit volume is zero. An unbalanced excess of positive charges creates a region of high electrical potential, repelling other positive charges and attracting negative charges. An excess of negative charges repels other negative charges and attracts positive charge. When an accumulation of positive charges on one side of a membrane is balanced by an equal and opposite accumulation of negative charges on the other side of the membrane, a difference of electrical potential (voltage) is set up between the two sides of the membrane.
Charge is carried back and forth across the cell membrane by small inorganic ions. These can traverse the lipid bilayer only by passing through special ion channels or pumps. When the ion channels open, the charge distribution shifts and the membrane potential changes. Of these ion channels, those whose permeability is regulated are the most significant; these are referred to as gated channels. Two classes of gated channels are of crucial importance: (1) voltage-gated channels, where the transmembrane potential controls the permeability of the channel; and (2) ligand gated channels, where the channel is opened (or closed) by binding of a chemical messenger to a receptor site.
Electrical activity in cells is generated by a complex interaction of both ligand and voltage gated ion channels. As a cell depolarizes (transmembrane potential changes), the voltage threshold for opening of different ion channels is reached, resulting in electrochemical gradient driven flow of ions through the cell membrane. Different channel classes open at different times and contribute to unique portions of this action potential. For example, a cardiac action potential can be divided into several phases: an initial rapid depolarization due to increased Na+ conductance; a plateau phase due to the slow opening of voltage-gated Ca++ channels; and the final repolarization due to the closure of Ca++ channels and the prolonged opening of K+ channels. Most, if not all, ion channels are modulated by signal transduction mechanisms that shape the action potential. For example, in the heart, six principal ion currents contribute to the nodal and pacemaking cell action potential and ten underlie the myocardial action potential.
Ion channels are multi-subunit, membrane bound proteins. In humans, ion channels comprise extended protein families with hundreds, or perhaps thousands, of both closely related and highly divergent family members. These channel superfamilies can be broadly classified into groups, based upon the specific ion, the type of gating, and the number of transmembrane domain segments and pore segments in the mature protein. The functional protein is a multimer that comprises one or more types of alpha subunits, and which are frequently associated with auxiliary, xcex2 subunits.
The effect of a compound on ion channels, and on the action potential of a living cell, can provide useful information about the classification and identity of the compound. Methods and means for extracting such information are of particular interest for the analysis of biologically active compounds, with specific applications in pharmaceutical screening, drug discovery, environmental monitoring, biowarfare detection and classification, and the like.
Relevant Literature
Examples of whole cell based biosensors are described in Gross et al. (1995) Biosensors and Bioelectronics 10:553-567; Hickman et al. (1994) Abstracts of Papers American Chemical Society 207(1-2):BTEC 76; and Israel et al. (1990) American Journal of Physiology:Heart and Circulatory Physiology 27(6):H1906-H1917.
Connolly et al. (1990) Biosens Bioelectron 5(3):223-34 describe a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes were tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained.
The molecular control of cardiac ion channels is reviewed by Clapham (1997) Heart Vessels Suppl 12:168-9. Oberg and Samuelsson (1981) J Electrocardiol 14:13942, perform fourier analysis on the repolarization phases of cardiac action potentials. Rasmussen et al. (1990) American Journal of Physiology 259:H370-H389, describe a mathematical model of electrophysiological activity in bullfrog atria.
Methods and apparatus are provided for the classification of biologically active molecules according to their effect on ion channels, changes in membrane potential, and the shape of action potentials that they evoke. A compound to be tested is put in contact with a biosensor, which has both living and non-living components. The living component comprises cells capable of excitation, which have a plurality of functional ion channels, and the non-living component comprises a means of monitoring changes in electric potential or ionic currents across the cell""s membrane. The test compound elicits a characteristic change in the shape and pattern of the membrane potential in the cell, over a range of frequencies.
The changes in electric potential or ionic current are recorded, and the data used to determine the spectral changes that are generated. In a preferred embodiment, the data is digitized and converted to a power spectral density (PSD). The spectral changes may be compared to those found with a control compound or condition, e.g. the absence of the compound. Unknown compounds may be identified by matching the characteristic spectral changes or pattern of spectral changes across a band of frequencies, as compared to a known reference pattern. Alternatively, unknown or known compounds, including derivatives and analogs of pharmaceutically active compounds, are classified as to general type, with respect to ion channel activation according to the particular pattern of responses that the compound evokes.
In one embodiment of the invention, the biosensor comprises cells grown on a microfabricated array having microelectrodes disposed over a substrate, with each microelectrode individually connected via conductive traces to contact points for electrical connection of external circuits.
In another embodiment of the invention, the biosensor comprises a panel of cells comprising different combinations of ion channels. A number of different panels are useful, including but not limited to panels having one type of cell from a variety of species, e.g. cardiac cells from multiple sources; different types of excitable cells from one or more species, e.g. neural, cardiac and sensory cells; and genetically engineered cells that either express ion channels not normally present in that cell type, or that lack expression of specific channels.